Q & A

September 2003

Q. What can we use as a positive control for a Heinz body prep test? What constitutes adequate proficiency testing? No one can remember the last time we had a positive result, but our hematologists want to retain the test as part of their hemolytic anemia workup.  

A. Heinz bodies are focal precipitates of oxidatively denatured hemoglobin within the red blood cell that are visible only with supravital dyes. Methyl violet, crystal violet, and brilliant green generally stain only Heinz bodies; reticulocyte stains, such as new methylene blue, can also be used to demonstrate Heinz bodies.

Heinz bodies appear as single or multiple irregular inclusion bodies adjacent to the cell membrane. Some Heinz bodies are formed in all people, but the spleen rapidly clears them. Increased numbers of Heinz bodies are seen in patients who are splenectomized; have decreased levels of red cell enzymes, such as individuals with glucose-6-phosphate dehydrogenase (G-6-PD) deficiency, especially if exposed to oxidant drugs; or have certain unstable hemoglobins, such as hemoglobin Zurich. Heinz bodies also form in anticoagulated blood incubated at room temperature for several hours and can be formed in vitro by exposing blood to oxidizers such as phenylhydrazine.

Valid Heinz body evaluations need the following:
• procedural controls—a sample run with the patient samples that will reliably demonstrate Heinz bodies to confirm that your staining procedure worked
• appropriate normal reference intervals for the types of patients you will be evaluating based on your specimen type and staining procedure
• appropriate proficiency testing.

As a procedural control, two alternatives are potentially viable. The first is to identify individuals who have been splenectomized or have Hb Zurich and who are willing to provide a sample using a protocol approved by your institution’s human protection program. This alternative is usually limited to very large institutions. The second is to use anticoagulated blood that exceeds what is needed for testing and has been retained at room temperature for a known duration. The latter obviously is an easier alternative, but it still requires appropriate treatment of the specimen.

An internally validated reference range is important because different staining protocols can themselves induce Heinz body formation. The age of the blood sample to be used is important. These ranges must also attempt to include all groups you are likely to test—neonates, for instance, have more Heinz bodies than older children and adults because they have developmentally lower levels of G-6-PD. Fortunately, individuals with true Heinz body anemias often demonstrate multiple Heinz bodies in more than 50 percent of cells, levels that far exceed those seen in healthy people.

Since no proficiency program offers Heinz body testing materials, laboratories must develop their own proficiency testing. It would be appropriate to send split samples to another certified lab that uses a procedure similar to that used by your lab or to conduct blinded testing of known samples.

Bibliography

• Heinz bodies. In: Howanitz JH, Howanitz PJ, eds. Laboratory Medicine. New York, NY: Churchill Livingstone; 1991: 479–480.
• Heinz body staining. In: Beutler E, Lichtman MA, Coller BS, et al, eds. Williams Hematology, 5th ed. New York, NY: McGraw-Hill; 1995:L26.

Robert Novak, MD
Department of Pathology
Children’s Hospital
Medical Center of Akron (Ohio)
Chairman, CAP Hematology/Clinical Microscopy Resource Committee

Q. What are some applications of tandem mass spectrometry, particularly for use in metabolic screening programs?

A. The application of mass spectrometry that has gained the most notoriety during the past 10 years is in the field of newborn screening. By the end of this year, more than one fourth of all infants born in the United States will have their filter paper blood specimens analyzed by mass spectrometry.

Among the metabolites analyzed are amino acid and acylcarnitine biomarkers indicative of more than 35 diseases. Other mass spectrometry applications in screening programs may include the analysis of homocysteine, 17-hydroxyprogesterone, bile acids, steroids, very long chain fatty acids, thyroxin, and other small molecules.

Although mass spectrometry can be used in protein analysis, especially hemoglobin and the characterization of sickle cell disorders, it has not been widely used in routine screening. Other mass spectrometers, such as time of flight, will likely be used in the next five to 10 years for protein analysis in screening programs. Furthermore, many new methods will be developed for use in high-risk analysis, including second-tier confirmatory tests.
The success of mass spectrometry applications in clinical screening has been a result of a single broad spectrum metabolite analysis for more than 50 biomarkers. The cost per analysis relative to the number of disorders detected is incredibly low. In fact, this same principle has produced a new quantitative method for measuring free carnitine in the plasma of dialysis patients, an area not specifically associated with metabolic screening programs.

Detailed reviews and discussions of mass spectrometry in clinical chemistry can be found in Chemical Reviews (Chace DH. 2001; 101: 445–477) and Annual Reviews of Genomics and Human Genetics (Chace DH, et al. 2002;3:17–45).
Donald H. Chace, PhD
Section Chief
Division of Bioanalytical
Chemistry and Mass Spectrometry
Pediatrix Screening
Bridgeville, Pa.